This proposal will focus on the tendon fibroblast surfaceome (the proteins on the tendon fibroblast surface) and its role in regulating collagen fibrillogenesis and extracellular matrix (ECM) assembly. Dysregulation of fibrillogenesis or assembly is associated with musculoskeletal disorders such as improper tendon formation, tendonitis and impaired tendon healing after injury. The general hypothesis of this proposal is that the tendon fibroblast surfaceome changes with development and the differing repertoire of proteins expressed regulates the stages of tendon formation. For aims 1 and 2, tendons from wild type PI mice will be used. At PI, tendons are predominantly in the earliest stage of formation (nucleation), where the fibroblast surface is crucial. Collagen V (Col V) and XI (Col XI) are expressed at the tendon fibroblast surface and interact there in the initiation of collagen fibril assembly. Aim 1 is to define the mechanism by which Col V and Col XI are associated with the fibroblast membrane and which specific proteins are responsible for tethering these collagens to the fibroblast membrane. Integrins and proteoglycans may be involved in the linkage of Col V and XI to the tendon fibroblast surface. The experimental design includes a Brij detergent extraction of the proteins from tendons, immunoprecipitation (IP) of Col V and XI and their associated proteins, and proteomic identification of these proteins. The data will be confirmed by localization of these candidate proteins in tendon extracts by (Western) blots and on the fibroblast surface by immunohistochemistry (IHC). Aim 2 is to identify the proteins that comprise the tendon fibroblast cell-surface and surface-associated (matrix) repertoire and that have roles in regulating tendon function. The surfaceome may also include molecules essential in signal transduction (such as FGF receptors), cell anchoring (such as adhesion molecules) and cell migration and matrix turnover (such as metalloproteinases). The proteins in the tendons will be extracted with Brij and identified by proteomics. Proteins identified in Aim 2 but not Aim 1 will be localized in extracts by blots and in tendons by IHC. Aim 3 is to define the developmental differences in the regulatory fibroblast surfaceome proteins identified in aims 1 and 2. Wild-type animals at 1-month (growing), 3-months (mature) and 18-months (aging) will be used. The candidate proteins from Aims 1 and 2 will be localized in tendon extracts from these animals by blots and in tendons by IHC. Knowing the regulatory proteins of developing tendons and their age-related changes allows better exploitation of the known regenerative capabilities of immature tendons. This will allow better clinical treatment and even reversal of debilitating tendon conditions in those with genetic tendon defects or who are aged or injured.